Exploration of Bacterial Re-Growth as In Vitro Phenomenon Affecting Methods for Analysis of the Antimicrobial Activity of Chimeric Bacteriophage Endolysins

Drug alternatives to combat methicillin-resistant Staphylococcus aureus (MRSA) in human and animal healthcare are urgently needed. Recently, the recombinant bacteriophage endolysins, PRF-119 and its successor substance HY-133, have proven to be highly active against various S. aureus clonal lineages and to exhibit a very rapid bactericidal effect when standard methods for susceptibility testing are applied. Along with subsequent growth curve experiments, a re-growth phenomenon was observed in vitro necessitating its clarification for the assessment of the agent’s stability and activity as well as for methodological aspects of endolysin testing in general. Distinct in vitro parameters were comparatively examined applying also scanning electron microscopy, fluorescence assays and SDS-PAGE analysis. The shape and material of the culture vessels as well as the shaking conditions were identified as factors influencing the in vitro stability and activity of HY-133. The highest function maintenance was observed in plain centrifuge tubes. Based on this, the conditions and parameters of assays for testing the antimicrobial activities of phage endolysins were determined and adjusted. In particular, shear forces should be kept to a minimum. Our results form the basis for both future test standardization and re-growth-independent experiments as prerequisites for exact determination of the antimicrobial activities of engineered endolysins.

Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of community-and healthcare-associated human and animal infections worldwide [1]. As it is a serious threat for individual patients and additional socioeconomic burden for healthcare systems, infection control measures including de-colonization strategies may help to prevent infections caused by healthcare-, community-and livestock-associated MRSA [2][3][4][5]. Increasing resistance towards topical agents for nasal MRSA de-colonization including mupirocin and biocides necessitates the search for alternative MRSA-active topical antibacterials [6][7][8][9]. Phages and, in particular, bacteriophage-derived lysins have recently been considered as an alternative therapeutic strategy to combat also the escalating problem of resistance in human and animal healthcare [10]. HY-133 (HYPharm GmbH, Bernried, Germany) is a chimeric bacteriophage endolysin, which may open novel strategies for nasal de-colonization of patients carrying S. aureus, in particular MRSA. Subsequent to the determination of the specific S. aureus activity of the predecessor agent PRF-119 [11], HY-133's activity against different clonal lineages and phenotypic variants of the S. aureus complex including livestock-associated MRSA, Staphylococcus schweitzeri strains, and different clinical and laboratory strains exhibiting the small-colony variant (SCV) phenotype was verified by means of standard methods for testing antimicrobial activities [12][13][14]. In a recent in-depth study, time-kill curves revealed a very rapid bactericidal effect of HY-133 even with low concentrations [15]. Unexpectedly, regrowth was observed after prolonged incubation times, the mechanisms of which remained largely unclear [15]. This phenomenon was also observed in experiments with graft surfaceadherent cells where HY-133 showed moderate bactericidal effect [16]. Here, we aimed to elucidate the nature of this re-growth phenomenon and to study in detail the in vitro test conditions that favor or prevent it.

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Determination
Minimum inhibitory concentration (MIC) of HY-133 against S. aureus strain ATCC 29213 was determined in sterile 96-well microplates (Greiner Bio One International, Kremsmünster, Austria) following the Clinical and Laboratory Standards Institute (CLSI) guidelines, as previously described [12,17,18]. Briefly, two-fold dilution series of HY-133 were prepared in cation-adjusted Mueller-Hinton broth (CAMHB, Becton Dickinson, Franklin Lakes, NJ, USA), with final concentrations ranging from 0.016-8 µg/mL. S. aureus ATCC 29213 was inoculated via direct colony suspension from stationary phase cultures grown overnight on Columbia blood agar (Becton Dickinson). A final concentration of 5 × 10 5 CFU/mL was used for inoculation of the 96-well microplates.
For determination of the minimum bactericidal concentration (MBC), 10 µL aliquots of culture from each microwell displaying the MIC and concentrations above were inoculated onto tryptic soy agar (TSA, Becton Dickinson) plates and incubated at 37 • C overnight. The MBC was defined as the concentration of HY-133 resulting in <500 CFU/mL (killing rate of 99.9%) [18]. Tests were performed in triplicate, and median values were taken for analysis.

Time-Kill Assays
Killing kinetics of HY-133 against S. aureus strain ATCC 29213 under different conditions were analyzed by means of time-kill curves following CLSI guideline M26-A [19]. In brief, cultures were grown on Columbia blood agar plates (Becton Dickinson) at 37 • C overnight. Several colonies were then inoculated in a pre-culture (10 mL tryptic soy broth, TSB, Becton Dickinson) and cultivated at 37 • C and 160 rpm. After 3 h, pre-cultures were adjusted in CAMHB (Becton Dickinson) to starting inocula of 5 × 10 5 CFU/mL. Time-kill kinetics were evaluated under different conditions as follows:

Scanning Electron Microscopy
To visualize the impact of different culture flask materials on HY-133's efficacy and structure, samples were prepared in the same way as described for time-kill assays in different culture vessels. Briefly, the 16-fold MIC of HY-133 was applied against S. aureus strain ATCC 29213 in (i) GBFs, (ii) PBFs, and (iii) CTs. Small pieces (0.5 × 0.5 cm) of the respective materials were added into the corresponding culture flasks during incubation. Cultures were kept for 3 h at 37 • C and 160 rpm. Subsequently, the small pieces of the different materials were removed from cultures and prepared for scanning electron microscopy (SEM) as previously described [20,21]. In brief, samples were fixed in 2.5% (v/v) glutaraldehyde in 0.1 M phosphate buffered saline (PBS) (pH 7.5) overnight followed by three times washing with PBS [22]. Samples were then post-fixed in 1% (w/v) osmium tetroxide in 0.1 M PBS (pH 7.5) and washed again in PBS (pH 7.5) for 3-5 min. Removal of water was accomplished by a graded water-ethanol series of 10-15 min steps (1 × 30%, 1 × 50%, 2 × 70%, 1 × 90%, and 2 × 100%). For high-resolution field-emission scanning electron microscopy (FESEM), samples underwent critical point drying using liquid CO 2 . Samples were attached to an aluminum carrier, then coated with 20 nm gold via sputter coating. Images were taken with an S-800 SEM (Hitachi Ltd., Tokyo, Japan) at room temperature and 20 kV acceleration voltage.

Fluorescence Assays
HY-133 protein activity under different conditions was determined using the HY-133 Activity Assay Kit (version 3.0, HYPharm GmbH) following the manufacturer's instructions. For this, HY-133 was suspended in PBS at a final concentration of 400 µg/mL. The suspension was aliquoted into GBFs, PBFs, CTs, and BCTs and incubated at 37 • C either with shaking (160 rpm) or stationary (0 rpm). Samples were drawn after 6 h, 24 h, and 48 h and used for activity measurements via fluorescence of a target substrate. The substrate is an oligopeptide containing the fluorescence dye N-methyl-anthraniloyl (N-Me-Abz) and a quencher (2,4-dinitrophenol, DNP) inhibiting the fluorescence signal when the peptide is intact. These components are linked with amino acids in the following order: DNP-L-Lys-(Gly)5-D-Ala-L-Lys-N-Me-Abz. This structure corresponds to the pentaglycine interpeptide bridge characteristic for the S. aureus peptidoglycan layer [23,24]. The active HY-133 cleaves the substrate at the cross bridge between Gly and D-Ala, leading to the fluorescence of the substrate via loss of the quencher function. The fluorescence signal was observed under stimulation and emission wavelengths of 340/30 nm and 440/40 nm, respectively (Synergy HTX Multi-Mode Microplate Reader, Biotek, Winooski, VT, USA). The specific activity (U/mg) was calculated by means of a calibration curve generated with a standard provided by the manufacturer (HYPharm GmbH). A positive and a negative control, provided in the kit, were included in each measurement. The experimental procedure was carried out in three biological replicates.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
HY-133 stability was evaluated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Samples were drawn and cleaned up with the ReadyPrepTM 2-D Cleanup Kit (Bio-Rad, Hercules, CA, USA) following the manufacturer's instructions. Samples were resuspended in 1× Laemmli sample buffer, incubated at 98 • C for 10 min, and then loaded on an 8-16% Mini-ProteanR TGX Stain-FreeTM Gel (Bio-Rad) at a final quantity of 45 µg each. Gels were stained in Coomassie brilliant blue overnight. Protein bands were visualized in the ChemiDoc Touch Imaging System (Bio-Rad).

Results
By microdilution, median MIC and MBC of each 0.5 µg/mL of HY-133 were determined for S. aureus ATCC 29213. Based on microdilution data, 1-, 2-, 4-, 16-, 32-, and 64-fold MICs of HY-133 were applied in time-kill experiments. The killing kinetics showed a decrease in CFU/mL within the first hour of the experiment for all concentrations of HY-133 applied. A minimum of the four-fold MIC induced a >3−log 10 reduction in CFU/mL in the first hour of the experiment, a minimum of the 16-fold MIC led to a decrease below the detection limit of <100 CFU/mL (  (Figure 2).
A potential loss of the in vitro activity of HY-133 over time was further analyzed via pre-incubation of the substance under the particular conditions of a time-kill experiment as previously established. For this, HY-133 was incubated for different time intervals in GBFs shaking at 160 rpm and tempered at 37 • C before S. aureus ATCC 29213 culture was added to the medium. HY-133's pre-incubation for 0.5 h led to a reduction in cell numbers <3 log 10 . Longer pre-incubation times of 1 h or 2 h, respectively, led to a complete loss of the enzyme's function, shown in growth curves similar to the curve of the growth control culture (Figure 3).   The causality of the environmental conditions in the time-kill curve experimental setting and the in vitro efficacy of HY-133 was further explored by analyzing the impact of the culture flask's form and material. For this, GBFs, GFs, PBFs, PFs, BCTs, and CTs (see Table 1) were utilized. For incubation at 160 rpm, the earliest re-growth of S. aureus strain ATCC 29213 was observed for GBFs, whereas cultures in GFs, PBFs, and PFs remained under the detection threshold (<100 CFU/mL) for a longer period of time. The most efficient killing of S. aureus cells was shown for the cultures in CTs. Here, no re-growth of the culture was observed over the experiment's time span of 48 h (Figure 4).
The impact of the flask material and shearing force on the stability and function of HY-133 was confirmed by analyzing the enzyme's specific activity under different conditions over time. For both baffled culture flasks, a strong reduction of HY-133's activity was observed after 24 h at 160 rpm. In the same culture flasks, the activity remained stable for 24 h, when incubation was carried out without shaking. The highest function maintenance for HY-133 over time was observed in plain CTs, where after 48 h the enzyme's function was still comparable to fresh HY-133 ( Figure 5A).
These results were further mirrored by SDS-PAGE analysis showing the stability of the protein over time. Here, for all approaches where HY-133 had been incubated shaking at 160 rpm and at 37 • C in baffled culture flasks, after 24 h, bands at expected height (32 kDa) became much weaker compared to those of the other approaches ( Figure 5B). Bands on the SDS gel remained much stronger for the samples when HY-133 was incubated in the same culture flasks at 37 • C but without shaking. This difference was apparent irrespective of the culture flask material. In contrast to this, when incubated in plain CTs, the stability of the protein was maintained over 48 h even when the tubes were shaken at 160 rpm ( Figure 5B).

Discussion
Today, we face the challenge of bringing new antimicrobials to the market. Optimal novel antimicrobial substances should bear the following characteristics: (i) be more refractory to resistance development; and (ii) be as specific as possible to a particular pathogen in order to protect the beneficial microbiota. Phage endolysins may address these requests and, thus, are promising candidates for alternative antibiotic substances. Their mode of action is independent of the metabolism of the bacteria, as they simply form holes in the bacterial cell wall through digestion of the peptidoglycan. Even if doses of endolysins were sub-lethal, a formation of resistance in the target bacteria would be unlikely, as the respective regions in the bacterial cell wall are highly conserved [25,26]. Furthermore, bacteriophage lysins tend to only aim at a certain bacterial species or subspecies [27]. Moreover, lysing bacterial cells within minutes, bacteriophage endolysins exhibit a very fast activity compared to classical antibiotics [15].
For the chimeric phage endolysins PRF-119 and the successor HY-133, rapid and specific action against S. aureus, mediated by the cell wall binding domain (i.e., lysostaphin), was already demonstrated in several studies [11][12][13][14][15]. For the precursor PRF-119, a "trailing effect" was described for microdilution assays, shown in point-like growth in wells with agent concentrations higher than the MIC [11]. Since this effect might have indicated a certain deficiency in stability of the protein structure, the stability-optimized HY-133 agent was designed showing no "trailing effect" in standard microdilution susceptibility assays. However, subsequent in-depth investigations of the in vitro activity of HY-133 in time-kill studies unexpectedly revealed re-growth of S. aureus strains, a phenomenon that had not occurred in previous microdilution susceptibility assays with HY-133 [11][12][13][14][15]. It is unlikely that the re-growth was caused by the development of resistance. In previous studies of our group, the survivor cells were susceptible to endolysin when sub-cultured and retested [15]. Thus, suboptimal standard in vitro conditions used for testing were concluded as a working hypothesis. Consequently, this study was intended to elucidate interfering assay conditions as a prerequisite for standardization of bacteriophage endolysin testing and as a methodological basis for future in vitro studies dealing with engineered bacteriophage products.
To test its susceptibility, the test strain ATCC 29213 was first subjected to the broth microdilution assay. It was found that both MIC and MBC of HY-133 for ATCC 29213 were generally low (0.5 µg/mL) and comparable to MICs and MBCs against other S. aureus strains tested before [13,14]. The identical values for MIC and MBC further proved an absence of phenotypic antimicrobial tolerance in strain ATCC 29213 [19,28]. In time-kill experiments, re-growth of strain ATCC 29213 was demonstrated, as already observed in the context of previous studies [12,15]. A 64-fold increase of the MIC or multiple doses of HY-133 at different time points during the experiment could not compensate for this effect. Via systematic pre-incubation of HY-133, we found that the enzyme completely lost its action against the test strain after only 1 h of incubation under the previously used test conditions. Shape and material of the culture vessel hitherto used for the experimental procedure were identified as causes for this loss of efficacy. The combination of unfavorable form and material was shown to lead to a loss of function of the enzyme when shaken at 160 rpm. The shaking of the culture flask, however, is an important factor in in vitro testing for (i) an adequate distribution of both substance and bacteria and (ii) an adequate supply of oxygen in the culture in order to provide ideal growth conditions for aerobic bacteria. By using a round vessel with a soft plastic surface, we were able to show that shaking at 160 rpm lost its negative impact on HY-133's in vitro activity completely.
The analysis on HY-133's functional activity further substantiated this finding. Here, HY-133 induced a stable fluorescence signal of the test substrate over the time span of 48 h when incubated in plain CTs, comparable to the enzyme's activity when freshly applied. This was also mirrored by demonstrating the stability of HY-133 applying SDS-PAGE analysis. Protein bands of HY-133 incubated in CTs at 160 rpm were comparable to those of the freshly tested endolysin. By contrast, baffled culture flasks led to a decrease in both activity and stability of HY-133 when incubated at the same speed. This decrease was observed for the different flask materials tested. However, the strongest effect was seen in GBFs, whereas the weakest effect was obvious in the BCTs. This is another hint that, apart from the culture flask form, also its material is decisive for the enzyme's activity. Similar to its precursor PRF-119, HY-133 consists of a cysteine-and histidine-dependent amidohydrolase/peptidase (CHAP) domain from the endolysin of phage K acting as enzymatic active domain (EAD) and a lysostaphin-based cell wall-binding domain (CBD) [11,14,[29][30][31]. The EAD and the CBD are connected through a linker peptide. Although already shortened and optimized compared to the linker in PRF-119, this part of the recombinant agent might be the weak part in in vitro tests applying relatively harsh environmental assay conditions for those linker peptide-containing proteins [11]. However, this does not necessarily imply instability of the agent in vivo.
SEM analysis underpinned the growth curve results. Applying different assay conditions, HY-133 activity resulted in the absence of intact S. aureus cells and formation of conglomerates consisting of the content of lysed bacterial cells and possible residues of inactive endolysin. These conglomerates also became macroscopically visible after a prolonged time span of 24 h (data not shown) and were mirrored by loss of protein bands in SDS-PAGE analysis. Besides these conglomerates, bacterial cell ghosts appear with empty irregular cell envelopes in PBFs and GBFs. These bacterial ghosts were detectable on the top as well as within the conglomerates. In contrast to this, in plain CTs, no cell ghosts were seen. Despite its pronounced in vitro susceptibility apparent in all culture flasks, it is probably that the highest proportion of active HY-133 could be maintained in the plain CTs leading to an efficient and complete cell wall destruction before formation of the conglomerates. Due to the ultra-fast mode of action of the endolysin, large amounts of the cell content of many S. aureus cells are simultaneously released and one could speculate that the resulting cell debris may enclose occasional a few S. aureus cells before being lysed. Thus, in vitro, HY-133 might be virtually "a victim of its own success" leading to protective effects which are not to be expected under in vivo conditions with lower bacterial cell count. Such a "protective effect by hiding" could be hypothesized in the case of PBFs and GBFs. This might also be the reason for the inefficiency of repeated doses of the endolysin at successive time points. In this case, the bacterial cells enclosed were not reached by the freshly added HY-133 anymore. The prolonged maintenance of HY-133 in plain CTs and the absence of ghost cells is also in line with the results of (i) the time-kill curves, (ii) the activity, and (iii) the stability assays. Accordingly, we highly recommend the utilization of plain CTs for killing kinetic analyses and other experiments in which high-speed shaking is necessary.

Conclusions
In summary, we were able to identify distinct factors impairing the in vitro stability and, hence, the in vitro activity of HY-133. By determining the influence of assay-related environmental conditions and material properties on the structure, stability and activity of this endolysin, we elucidated optimized conditions for in vitro testing of HY-133. In particular, shear forces should be kept as low as possible. If shaking is unavoidable, tubes with a plain surface should be used. These findings should be considered for future attempts to establish standard recommendations for testing and analyzing the in vitro activities of bacteriophage endolysins, in particular for those with chimeric structure. The observed protective effect on very few S. aureus cells, probably associated with extremely rapid "overkilling", could explain the phenomenon of re-growth. Although most likely limited to in vitro conditions, this warrants further investigation and should be considered for future treatment strategies with bacteriophage endolysins.


CBP/p300 activation promotes axon growth, sprouting, and synaptic plasticity in chronic experimental spinal cord injury with severe disability

The interruption of spinal circuitry following spinal cord injury (SCI) disrupts neural activity and is followed by a failure to mount an effective regenerative response resulting in permanent neurological disability. Functional recovery requires the enhancement of axonal and synaptic plasticity of spared as well as injured fibres, which need to sprout and/or regenerate to form new connections. Here, we have investigated whether the epigenetic stimulation of the regenerative gene expression program can overcome the current inability to promote neurological recovery in chronic SCI with severe disability. We delivered the CBP/p300 activator CSP-TTK21 or vehicle CSP weekly between week 12 and 22 following a transection model of SCI in mice housed in an enriched environment. Data analysis showed that CSP-TTK21 enhanced classical regenerative signalling in dorsal root ganglia sensory but not cortical motor neurons, stimulated motor and sensory axon growth, sprouting, and synaptic plasticity, but failed to promote neurological sensorimotor recovery. This work provides direct evidence that clinically suitable pharmacological CBP/p300 activation can promote the expression of regeneration-associated genes and axonal growth in a chronic SCI with severe neurological disability.

Introduction
Spinal cord injury (SCI) is a devastating disease affecting millions of people worldwide. Severe SCI leads to permanent motor, sensory, and autonomic dysfunction that disrupts the quality of life of affected people. The management of severe SCI is nowadays limited to supportive care. Current physical rehabilitation has measurable but limited benefits after moderate SCI when these housing conditions are compared to patients, suggesting that EE should be used as "standard" housing condition. The lack of integration between approaches aimed to promote regenerative molecular mechanisms with neurorehabilitation and neuromodulation after SCI remains a major limitation for repair in severe and chronic SCI, where reawakening a regenerative gene expression programme and stimulation of disrupted neural activity are especially challenging and potentially critical to repair and recovery. Additionally, mechanistic and therapeutic advances in chronic SCI with severe disability are especially rare and therefore represent a high priority.

Hypothesis and relevance
Here, we hypothesize that the pharmacological stimulation of CBP/p300 activity will enhance regenerative gene expression during a growth refractory phase 12 weeks after spinal injury, while housing animals in an EE 1 week postinjury will stimulate neuronal activity, consolidate axonal and synaptic plasticity as opposed to animals housed in SH. Combined proteomics and transcriptomics studies indicate that EE activates physiological responses that are independent from CBP pathways as recently reported [21]. They include modulation of mitochondrial metabolism, calcium signalling, ion channels, axonal transport, and release of extracellular signalling vesicles among others, potentially allowing for synergistic benefits between the drug and EE.
Therefore, we postulate that CBP/p300 activator CSP-TTK21 on an EE housing baseline following the most clinically relevant chronic spinal cord injuries with severe disability might enhance neuronal plasticity, regeneration, and functional recovery, providing a better understanding of regenerative failure and filling a gap in the path to translation. Importantly, EE does not represent a specific form of focused rehabilitation, but rather a more physiological setting compared to SH that better reflects the human condition where patients are encouraged to engage in physical activities after a SCI. This explains the rationale for having animals in EE as baseline as opposed to having them in SH, which represents an impoverished artificial environment. Indeed, our study will test the effect of the CBP activator on this more "physiological" background in chronic SCI with severe disability.
The long-ranging implications of this work lie on testing a novel strategy based upon the CBP/p300 pharmacological activation to promote functional recovery after chronic SCI with severe disability, delivering the necessary preclinical evidence to support future clinical translation. Importantly, lack of validation of this hypothesis will also provide essential information allowing the scientific community to entertain alternative hypotheses. They include the possibility (i) that increases in regenerative gene expression and neural activity might not be synergistic in providing growth and plasticity; (ii) that recovery and repair might need task-specific neurorehabilitation; (iii) that reestablishment of functional circuitry needs targeting the spinal synapses; and finally (iv) that identification and manipulation of the extraneuronal wound healing and scarring processes in chronic SCI might need to be engaged along with favouring neuroplasticity.

Experimental design
The experimental design asked the question of whether activation of CBP and the cognate protein p300 with the weekly systemic delivery of TTK21 would enhance gene expression that supports plasticity and regenerative growth after chronic spinal cord transection injury with severe disability. Since no data were available on TTK21 in chronic SCI, a transection model in mice has been chosen versus a contusion in rats because (i) it allows a more accurate anatomical definition of axonal regeneration and sprouting; (ii) it has much less variability allowing for a more reliable statistical assessment keeping the number of animals relatively limited; and (iii) it needs a fraction of the drug required (in mice versus rats) that is produced in JNCASR, Bengaluru, India. Currently, the availability of the compound does not allow to initiate studies in rats in the short term; however, these studies will surely prompt further investigation in severe chronic contusion in rats, which is the next step towards human translation.
Specifically, adult 6-to 8-week-old C57Bl/6 mice received a spinal cord T9 transection injury that destroys the ascending sensory fibres in the dorsal columns bilaterally as well as most of the descending corticospinal, raphespinal, rubrospinal, and reticulospinal motor tracts, mimicking severe clinical SCI. This lesion leads to permanent severe impairment in sensorimotor function, severely limiting stepping up to at least 42 days postinjury, resembling clinical severe SCI (S1 Fig). All mice were placed in an EE 1 week after the SCI. The CBP activator TTK21 is coupled to slow-release carbon nanoparticles (CSP-TTK21) that allows a weekly administration maintaining stable activity levels in target tissues. Thus, CSP-TTK21 was administered via IP injections once per week starting 12 weeks after injury for 10 weeks. A control group of mice was treated with nanoparticles and vehicle alone (CSP). The CSP-TTK21 and control CSP nanoparticles were used at the dosage of 20 mg/kg that showed efficacy in subacute SCI as recently published [21].
AnimalsAU : PerPLOSstyle; donotusesacrificeinreferencetokillingofanimalsduringexperiments:Usek were killed at week 22 postinjury. Sprouting and regeneration of the dorsal columns were analyzed with the retrograde axonal tracer Dextran-488 (injected in the sciatic nerve in proximity of L4-L6 dorsal root ganglia (DRGsAU : PleasenotethatDRGshasbeenfullyspelledasd ) 5 to 7 days before killing the animals). This allows visualization of ascending fibres in the dorsal columns into and across the injury site as previously shown in Di Giovanni's lab [21]. Sprouting and regeneration of the corticospinal tracts (CSTs) were analysed with stereotaxic injections of the neural tracer Dextrantetramethylrhodamine and biotin (Dextran T&B), into the motor cortex 2 weeks before killing, which allows visualization of descending fibres into and across the injury site according to standard procedures in Di Giovanni's lab [24]. Axonal dieback as well as the number of fibres past the lesion site were normalized to the number of labelled fibres prior to the lesion as previously shown [24]. Given it closely correlates to locomotion, we also measured sprouting of serotoninergic raphe-spinal motor tracts with 5-HT immunohistochemistry as recently described [21]. To assess whether the CSP-TTK21 treatment enhances synaptic plasticity, we measured the number of inhibitory vGat or excitatory vGlut1 synaptic terminals in proximity of neuronal targets such as interneurons in the dorsal horns and motoneurons in the ventral horns of the spinal cord (ChAT or NeuN immunostaining) including in association with specific tracing of CST, sensory, or 5-HT fibres. This was carried out by fluorescent multilabelling experiments that were analyzed by confocal microscopy. Histone acetylation as read out of CBP/p300 activation was also evaluated in layer V neurons, raphe nuclei, and DRG neurons by immunofluorescence. The expression of several regeneration associated factors including ATF3, JUN, GAP43, SPRR1a, KLF7, pERK, and pSTAT3 was also studied by immunofluorescence in sensory and motor neurons.
In addition, we assessed locomotion, coordination, and sensorimotor integration by performing open field assessment with the Basso Mouse Scale (BMS) and the gridwalk tests, as recently shown [21]. Lastly, Von Frey test for mechanoception and mechanical allodynia as well as Hargreaves test for thermoception and thermal hyperalgesia were used to specifically assess the function of the ascending sensory tracts.
Please find a graphical summary (S2 Fig) and a summary table (Table 1) of the experimental design.

CBP/p300 activator TTK21 enables axonal growth and synaptic plasticity in chronic SCI
Delivery of the CBP/p300 activator TTK21 significantly reduced axonal retraction and promoted axonal growth of motor corticospinal (Fig 3A and 2B) and of sensory DRG neurons, respectively (Fig 3C and 3D). However, the most striking phenotype was the increased sprouting of 5-HT raphe-spinal axons as shown by 5-HT immunofluorescence (Fig 3E and 3F). Importantly, TTK21 led to an increased number of vGlut1, but not of vGat, boutons opposed to motor neurons in the ventral horn of L1-3 spinal sections below the injury site (Fig 3G-3L).
No difference in the size of the lesion and in GFAP astrocytic immunostaining was observed between the experimental groups (S4A- S4C Fig). Similarly, measurement of macrophage/microglia CD68 immunofluorescence intensity around the injury did not show any difference between vehicle and TTK21 (S5 Fig), suggesting that systemic activation of CBP/p300 does not lead to obvious changes in the spinal cord astrocytic and macrophage/microglia responses. Thus, CBP/p300 activation can promote axonal growth and synaptic plasticity in chronic SCI with severe disability.

CBP/p300 activator TTK21 does not affect neurological recovery in chronic SCI
Lastly, we measured whether CBP/p300 activation promotes sensorimotor recovery in chronic SCI. We did not observe any significant difference in the sensorimotor performance by BMS ( Fig 4A) and gridwalk (Fig 4B) between TTK21 and vehicle-treated animals, while the severity of the lesion was confirmed by the severe impairment observed (Fig 4C). Only a minority of animals were able to perform stepping on a gridwalk (BMS >3; Fig 4B). Similarly, the animals that could be tested for mechanoception by Von Frey and thermal responses by Hargreaves showed severe impairments with no difference between the 2 experimental groups (Fig 4D  and 4E). Thus, CBP/p300 activation cannot stimulate sensory or motor recovery with this level of chronic SCI.

Discussion
Chronic severe SCI that is associated with absent axonal regrowth and reconnectivity and severe neurological disability remains a major medical challenge. It is believed that the ability for axonal growth and sprouting declines over time after an SCI and that the time window for intervention might eventually close. The importance of our findings lies on the evidence that starting a treatment such as the CBP/p300 activator TTK21 12 weeks after an SCI in the mouse with severe disability can still elicit a regenerative response as shown by increased axonal growth, sprouting, and synaptic plasticity at 22 weeks postinjury. The CBP/p300 activator TTK21 also promotes the expression of regenerative signals including some well-established RAGs. This is a potentially exciting discovery since it provides a demonstration that a clinically suitable molecular intervention can promote plasticity and growth in both an acute, as previously shown [21], and chronic SCI with severe disability, likely by reawakening a dormant regenerative gene expression programme. While both motor and sensory neurons showed increased histone acetylation, CBP/p300 activation induced the expression of most RAGs investigated in DRG neurons only. These findings are in line with previous work in subacute SCI where these classical RAGs were not activated in the corticospinal [25], which temporarily revert to an embryonic state, as opposed to DRG neurons, which reexpress RAGs, as shown here. However, the increase in histone acetylation seems to be a common denominator of TTK21 delivery for sensory and motor neurons alike. Increased acetylation likely stimulates distinct regenerative programmes between peripheral sensory and central motor neurons by enhancing chromatin accessibility at gene regulatory regions of RAGs for sensory neurons and perhaps of developmentally regulated genes for corticospinal neurons as recent work from the Tuszynski's lab might suggest [25]. They found that reexpression of embryonic genes after SCI Mean ± SEM; unpaired two-tailed Student or Welch t test; � p < 0.05, ��� p < 0.001, ���� p < 0.0001. n = 3 or 4 biologically independent animals. The data can be found in S1 Data. CSP, carbon nanosphere; DRG, dorsal root ganglion; SCI, spinal cord injury; TOST, two one-sided tests. lasts for 2 weeks only in corticospinal neurons. In the present chronic state of injury (12 to 22 weeks after injury), this "primed" state for regeneration has likely closed. Thus, delivery of CSP-TTK21 might have partially reopened this regenerative window. ItAU : Pleasecheckandconfirmtha is in fact important that TTK21 increases the growth and regenerative gene expression ability of both sensory and motor neurons, albeit with differential potency and efficacy, being maximal for regenerative gene expression at selected RAGs in sensory DRG neurons and for axonal sprouting in 5-HT motor neurons. The very distinct embryonic origin and molecular identities of these CNS neuronal subpopulations might underline this differential response [26]. However, the variable distance from the lesion site and the rate of axonal transport of selected neuronal populations might be additional contributing factors since neuronal cell types and the distance of the neuronal cell body from the injury site affect the rate and extent of axonal trafficking and the expression of RAGs.
Given the severity of the spinal injury in the present study, it is very difficult to directly compare these findings with the level of axonal growth, sprouting, and synaptic plasticity observed in our previous work when we delivered TTK21 6 or 24 hours after a mouse spinal cord hemisection or a rat moderate to severe contusion injury, respectively [21]. However, the most striking difference between the present and the previous experiments is the lack of any neurological recovery. The most immediate explanation might be the modest effect of axonal growth of sensory and corticospinal neurons, the depth of the transection injury in the present study compared to a dorsal hemisection or a moderate to severe contusion in the previous. The presence of astrocytic rich tissue bridges is important to allow for axonal growth, and the present chronic injury showed poor tissue bridges. Additionally, the diverse injury severities and timing postinjury likely reflect a different glial environment that might influence synaptic transmission, repair, and functional recovery. Finally, in a chronic condition, synaptic transmission might be compromised by the functional impairment of neuronal targets below (motor targets) or above (sensory targets) the injury site due to the long-standing deafferentation.
However, our positive neuroanatomical and molecular findings despite the limitations of the lack of neurological recover might pave the way for the future combinatorial use of TTK21 . All data are given as mean ± SEM; unpaired two-tailed Student t test; � p < 0.05, ��� p < 0.001, ���� p < 0.0001. n = biologically independent animals. The data can be found in S1 Data. CSP, carbon nanosphere; CST, corticospinal tract; SCI, spinal cord injury; TOST, two one-sided tests.
https://doi.org/10.1371/journal.pbio.3001310.g003 activation with stem cell grafts [25,27], self-assembling peptide-based biomaterials [28], or both, since these might provide the necessary tissue bridge and relays to allow for increased functional synapses underpinning neurological recovery. Additionally, while in our view, tissue bridges will need to be part of the equation for spinal repair, the synergism with additional neuronal extrinsic interventions aimed to further enhance plasticity such as CSPG inhibition could also be considered.
More broadly, extending upon previous findings showing that PTEN deletion [29] or antagonism [30] benefit axonal growth and spinal circuitry formation in chronic SCI, the present work suggests that it is possible to promote axonal growth and plasticity in a chronic spinal cord injury leading to severe disability. Finally, similarly to TTK21, it encourages to attempt other molecular regenerative interventions, ideally in combination with tissue bridging approaches.

Mice
Mouse experimentation was carried out in accordance with regulations of the UK Home Office under the Animals (Scientific Procedures) Act 1986, with Local Ethical Review by the Imperial College London Animal Welfare and Ethical Review Body Standing Committee (AWERB, PPL P6EDD65B1). Female C57Bl6 (Harlan, UK) mice ranging from 6 to 8 weeks of age were used for all experiments. For all surgeries, mice were anesthetized with isoflurane (4% induction 2% maintenance) and buprenorphine (0.1 mg/kg) and carprofen (5 mg/kg) were administered peri-operatively as analgesic.
Animals were kept on a 12-hour light/dark cycle with food and water provided ad libitum, at a constant room temperature and humidity (21˚C and 5%, respectively). SH for mice consists of 26 × 12 × 18 cm 3 cages housing 4 mice with tissue paper for bedding, a tunnel, and a wooden chew stick. The EE housing consists of 36 × 18 × 25 cm 3 cages housing 5 mice with tissue paper for bedding, a tunnel, and a wooden chew stick. EE cages also host the following: additional nesting material that included nestlets, rodent roll, and sizzle pet (LBS Biotech); a hanging plastic tunnel (LBS Biotech) and a plastic igloo combined with a fast-track running wheel (LBS Biotech); a wooden object (cube, labyrinth, tunnel, corner 15) (LBS Biotech) that is changed every 5 days to help maintain a novel environment; and 15 g fruity gems (LBS Biotech) every 5 days to encourage exploratory and natural foraging behaviour.

Spinal cord injury (SCI)
Surgeries were performed as we previously reported [24]. A laminectomy at vertebra T9 was performed to expose spinal level T9 and a deep dorsal transection past the central canal at a depth of approximately 1.1 mm was carried out using microscissors (Fine Science Tools). This lesion only spares a portion of the ventral white matter and leaves the animals with a severe and permanent neurological impairment-no improvement from baseline. At week 22 after SCI, animals were deeply anesthetized and perfused transcardially.

CSP-TTK21 administration
Animals were randomised to treatment after been subdivided into 2 groups with comparable severity based upon BMS with the CBP/p300 activator bound to carbon nanospheres (CSP-TTK21) or a control of just CSP (mice-20 mg/kg injected IP once a week). Mice received the first IP injection 12 weeks after SCI until killing.

Neuronal tracing
For dorsal column tracing of sensory DRG axons, 2 μl of Dextran-88 (Life Technology) was injected into the sciatic nerve bilaterally 1 week before killing using the fire polished glass capillary connected to a 10-μl Hamilton syringe.
For anterograde tracing of motor CSTs, 2 weeks before killing, mice that received an SCI were injected with the axonal tracer Dextran-tetramethylrhodamine and biotin, with a Hamilton microsyringe in the motor cortex following standard stereotaxic coordinates as previously described [31].

5-HT immunostaining
For 5-HT immunostaining, we followed a protocol we previously described [32]. The sections were incubated with rabbit anti-5-HT (1:500, Sigma S5545) in 4% NGS in 0.3% TBS-Triton X100 for 4 days at 4˚C. Next, the sections were incubated with an Alexa fluorescent secondary antibody. Finally, the sections were coverslipped.

Behavioral analysis
Mice were trained daily for 2 weeks preinjury before baseline measurements and then assessed on day 7 postinjury and biweekly thereafter; until CSP or CSP-TTK21 injections, mice were then tested weekly thereafter. All behavioral testing and analysis were done by 2 observers blinded to the experimental groups.
Gridwalk. Mice will cross a 1-m long horizontal grid 3 times. Videos of the runs were blindly analyzed at a later time point, and errors from both hind limbs were counted and normalized to total number of steps. Error values represent the total number of slips made by both hindlimbs over the 3 runs.
Open-field test. The BMS [33] was used to assess open-field locomotion. Each animal was allowed to freely move in the open field for 4 minutes while 2 independent investigators blinded to experimental group will score it. The BMS score and subscore were given. Only the animals showing frequent or consistent plantar stepping in the open field (BMS score �3) were tested on the grid walk.
Von Frey. The Von Frey test determines the mechanical force required to elicit a paw withdrawal response. Each animal was tested in each paw 3 times. Only animals showing plantar placement in the open field (BMS score �3) were tested.
Hargreaves. The Hargreaves test determines the latency of a thermal nociceptive stimulus required to elicit a paw withdrawal response. Each animal was in each paw 3 times. Only animals showing plantar replacement in the open field (BMS score �3) were tested.

Quantification of axonal regeneration
For each spinal cord after injury, the number of fibres rostral and caudal to the lesion and their distance from the lesion epicentre (depending on whether sensory or motor axons) were analysed in 4 to 6 sections per animal with a fluorescence Axioplan 2 (Zeiss) microscope and with the software Stereo-Investigator 7 (MBF Bioscience). The lesion epicentre was identified by GFAP staining in each section at 20× magnification. The total number of labelled axons or signal intensity of the traced axons rostral to the lesion site were normalized to the total number of labelled axons or of the signal intensity caudal or rostral to the lesion site counted in all the analysed sections for each animal, obtaining an inter-animal comparable ratio. Sprouts and regrowing fibres were defined following the anatomical criteria reported by Steward and colleagues [34].

Histology and immunohistochemistry
Tissue was postfixed in 4% paraformaldehyde (PFA) (Sigma) and transferred to 30% sucrose (Sigma) for 5 days for cryoprotection, the tissue was then be embedded in OCT compound (Tissue-Tek) and frozen at −80˚C. DRG, spinal cord, and cortices were sectioned at 10, 20, and 20 μm thickness, respectively, using a cryostat (Leica). Immunohistochemistry on tissue sections was performed according to standard procedures. For selected antibodies, antigen retrieval was performed submerging the tissue sections in 10 mM citrate buffer (pH 6.2) or 10 mM Tris/1 mM EDTA buffer (pH 9.0) at 98˚C for 5 minutes. Next, tissue sections were washed with PBS to remove the excess of citrate buffer and blocked for 1 hour with 8% BSA, 1% PBS-TX100.

Image analysis for IHC
All analysis was performed by the same experimenter who was blinded to the experimental groups. Photomicrographs were taken with a Nikon Eclipse TE2000 microscope with an opti-MOS scMOS camera using 10× or 20× magnification using ImageJ (Fiji 64 bits 1.52 p), Micro-Manager 2.0 software for image acquisition or at 20× magnification with an Axioplan 2 (Zeiss) microscope and processed with the software AxioVision (Zeiss).

Analysis of GFAP and CD68 intensity around the lesion site
GFAP and CD68 intensity and area with positive signal were quantified from sagittal spinal cord sections from 1 series of tissue for each animal. Quantification was done using ImageJ, the background was subtracted, and then the mean pixel intensity and area of immunoreactivity was measured.

Analysis of fluorescence intensity
For quantitative analysis of pixel intensity, the nucleus or soma of DRG or layer V cortical neurons were manually outlined in images from 1 series of stained tissue for each mouse. To minimize variability between images, pixel intensity was normalized to an unstained area and the exposure time and microscope setting were fixed throughout the acquisition.

Analysis of 5HT fibres in the ventral horn
Intensity of 5HT immunohistochemistry was measured in the ventral horn of L1-4 spinal sections. Quantification was done using ImageJ, the background was subtracted, and then the mean pixel intensity was measured from 1 series of tissue for each animal.

Analysis of vGlut1 and vGat immunohistochemistry in proximity to motor neurons
vGlut1 and vGat synaptic boutons were imaged with a SP8 Leica confocal microscope. Zstacks images were taken with an average thickness of 15 μm with a step size of 0.3 μm. Sequential line scanning was performed when more than 2 channels were acquired. Multifluorescent orthogonal 3D image analysis and visualization were performed using Leica LAS X software. The average number of vGlut1 or vGat boutons opposed to motor neurons in the ventral horn of L1-3 spinal sections was calculated by analysing 20 motor neurons per replicate. All analyses were performed blind to the experimental group.

Statistical analysis
Measures for avoidance of bias (e.g., blinding, randomisation). We adhere to the principles of NC3Rs and adhere to the ARRIVE guidelines [35]. All experiments were performed in blind to the treatment and experimental group. Behavioral studies were assessed by 2 independent investigators (e.g., research associate and research assistant) blind to one another, to the treatment, and to injury group when relevant. Randomization followed a computerized sequence. As far statistical analysis, our priority was to adopt a frequentist equivalence test. There are 5 parameters in our experimental design: sample size (n), difference between the groups (delta), standard deviation (sigma), type I error (alpha/significance), and type II error (power). We can use estimates for 4 of these parameters to calculate the 5th parameter-which we have done to achieve our required sample size to achieve a certain power and significance.
Sample size calculation, power calculations, and justification of effect size. The size of our in vivo experimental groups as planned in our aims has been defined following the Animal Experimentation Sample Size Calculator (AESSC) tool. Sample size calculations were performed using a two-tailed unpaired Student t test and two-way repeated measures ANOVA (significance � 0.05; power � 0.90; G � Power); N indicates number/group. The specific effect size has been estimated based upon similar studies showing significant differences between experimental and control group [8,21].
Two animals were added to each experiment based upon the probability of losing animals due to the experimental procedure such as spinal surgery. While the selected N is derived by our power calculation, it is also compatible and comparable with what we have previously published for similar experiments [21]. Exclusion criteria include the following: • Tissues with low quality for further experimentation or imaging as shown by lack of clear injury site or by lack of clear axonal tracing. Degraded cords in case of inefficient fixation or damaged during cryostat sectioning.
• Animals with injury severity that differs 2 SD from the mean as defined by the size of the injury site, which has to be within 2 SDs from the average.
No animal replacement was needed to ensure that the power requirements were met. Specific calculations are found here below: Immunofluorescence (control vs. TTK21): change in fluorescence intensity of 1.5-fold (between 50 and 75 arbitrary units for example); SD = 10 and 12; P: 0.05; Power 90%; N = 4 Axonal regeneration (control vs. TTK21): increase in % of axons regenerating beyond the lesion site from 5 to 10; SD = 2 and 4; Power 90%; P: 0.05, N: 8 Behavioral tests (gridwalk as example test, control vs. TTK21): changes in score from 4 to 2; SD: 2 and 1; Power 90%; P: 0.05; N = 12 Results were expressed as mean ± SEM. Statistical analysis was carried out using GraphPad Prism 9. Normality was tested for using the Shapiro-Wilk test. Normally distributed data were evaluated using a two-tailed unpaired Student t test or a two-way repeated measures ANOVA when experiments contained more than 2 groups. The Tukey or Sidak post hoc test was applied when appropriate. The Mann-Whitney U test was used for nonparametric evaluation. Given the level of nonsignificance in behavioural experiments, a frequentist equivalence test using the two one-sided tests (TOST) rule was needed to strengthen the null hypothesis conclusion (TOSTER 0.4.1, parameters 0.18.1.6, R 4.1.2).
A threshold level of significance α was set at P value <0.05. Significance levels were defined as follows: � P value < 0.05; �� P value < 0.01; ��� P value < 0.001, ���� P value < 0.0001. All data analyses were performed blind to the experimental group.
URL to this deposited stage 1 manuscript: 10.17605/OSF.IO/S5EDH Supporting information S1 Fig. (A) Twelve weeks old mice were housed in SH or EE 1 week after a spinal cord transection. (B and C) Animals in SH remained impaired unable to step until day 42 after injury as shown by BMS (B) and Gridwalk (C). EE significantly enhanced locomotion (mean ± SEM, two-way ANOVA, Fisher LSD post hoc �� P < 0.01; ��� P < 0.005; ���� P < 0.001). The data can be found in S1 Data. BMSAU : AbbreviationlistshavebeencompiledforthoseusedinS1andS3 À S5Fig , Basso Mouse Scale; EE, enriched environment; SH, standard housing. (PDF)


Global Noncoding microRNA Profiling in Mice Infected with Partial Human Mouth Microbes (PAHMM) Using an Ecological Time-Sequential Polybacterial Periodontal Infection (ETSPPI) Model Reveals Sex-Specific Differential microRNA Expression

Periodontitis (PD) is a polymicrobial dysbiotic immuno-inflammatory disease. It is more prevalent in males and has poorly understood pathogenic molecular mechanisms. Our primary objective was to characterize alterations in sex-specific microRNA (miRNA, miR) after periodontal bacterial infection. Using partial human mouth microbes (PAHMM) (Streptococcus gordonii, Fusobacterium nucleatum, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia) in an ecological time-sequential polybacterial periodontal infection (ETSPPI) mouse model, we evaluated differential mandibular miRNA profiles by using high-throughput Nanostring nCounter® miRNA expression panels. All PAHMM mice showed bacterial colonization (100%) in the gingival surface, an increase in alveolar bone resorption (p < 0.0001), and the induction of a specific immunoglobin G antibody immune response (p < 0.001). Sex-specific differences in distal organ bacterial dissemination were observed in the heart (82% male vs. 28% female) and lungs (2% male vs. 68% female). Moreover, sex-specific differential expression (DE) of miRNA was identified in PAHMM mice. Out of 378 differentially expressed miRNAs, we identified seven miRNAs (miR-9, miR-148a, miR-669a, miR-199a-3p, miR-1274a, miR-377, and miR-690) in both sexes that may be implicated in the pathogenesis of periodontitis. A strong relationship was found between male-specific miR-377 upregulation and bacterial dissemination to the heart. This study demonstrates sex-specific differences in bacterial dissemination and in miRNA differential expression. A novel PAHMM mouse and ETSPPI model that replicates human pathobiology can be used to identify miRNA biomarkers in periodontitis.

Introduction
MicroRNAs (miRNAs) are small, noncoding, regulatory RNAs that play a major role in immune inflammation-mediated periodontal disease (PD). miRNAs are~22 nucleotides in length and regulate the expression of many messenger RNAs (mRNAs) by directly binding to their 3 untranslated regions [1]. Prior in vitro and in vivo studies from our laboratory [2] and others have shown that miR-146a [1,3,4], miR-30e, miR-106b [5], and miR-155 [6][7][8]  pathogenesis. Both innate and adaptive immune systems are affected by miRNA; therefore, studying the role of miRNA in inflammation-mediated periodontitis aids in developing prognostic/diagnostic biomarkers. PD affects > 20% of the world's population, and its prevalence varies between men and women [9]. These observed differences between sexes are likely due to the influence of sex hormones on periodontal inflammation and alveolar bone metabolism [10], sexual dimorphism in periapical inflammation and alveolar bone loss, differences in bone turnover and lymphocyte recruitment [11], and differences in the jaw as it grows, develops abnormalities, and ages [12,13]. Furthermore, men have a stronger inflammatory response to bacterial infection and greater osteoclast-driven alveolar bone resorption (ABR) than women [14]. Moreover, a recent study also reported sex-specific differences in the oral microbiome in patients with severe periodontitis [15].
Prior research has reported the differential expression (DE) of specific miRNAs in a PD animal model [2]. However, this study is the first to evaluate the DE of miRNA in a more clinically relevant ETSPPI mouse model stratified by sex and using an unbiased global high-throughput miRNA analysis. No single microbe is responsible for the progression of periodontitis in humans; microbes are linked with synergistic (physiological, nutritional, and biochemical) sequential interspecies and interkingdom interactions of several microbes, including the early, intermediate, and late bacterial colonizers of the periodontium. Several studies report that P. gingivalis (Pg), T. forsythia (Tf ), T. denticola (Td), and F. nucleatum (Fn) (coaggregating bacteria) are identified more frequently and in higher numbers in adults with periodontitis than in healthy individuals, and they are also positively correlated with pocket depth and bleeding on probing, which are measurements of periodontal tissue destruction [16]. Streptococcus gordonii (Sg), an early colonizer of dental plaque, coaggregates with many oral species, including intermediate colonizer F. nucleatum [17] and late colonizer P. gingivalis [18]. The coadhesion of P. gingivalis to S. gordonii is a key point in the pathogenesis of PD [18,19]. F. nucleatum strongly bridges Gram-positive (Gram+) and Gram-negative (Gram−) bacteria and assists other periodontal bacteria through the enhanced adhesion and invasion of host cells [20,21]. Accordingly, Sg+Fn+Pg/Td/Tf PAHMM and ETSPPI have a synergistic pathogenicity.
Here, we designed and characterized an ETSPPI mouse model using five bacteria Sg+Fn+Pg/Td/Tf in C57BL/6J mice (male and female). In addition, we used highthroughput miRNA expression profiling, a recent innovation, using the Nanostring nCounter ® system to study the global DE of miRNA in PAHMM-infected mice. We identified several significant sex-specific alterations in gingival tissue miRNAs after ETSPPI. Five of the miRNAs, namely miR-377, miR-690, miR-1274a, miR-669a, and miR-199A-3p, are reported here for the first time in mice. The other two miRNAs, miR-9 and miR-148a, have been shown to be expressed in inflamed human gingival tissues [1,6,22]. We also report bacterial colonization in the oral surface of mice after each reinfection and measure horizontal ABR and the immunoglobulin G (IgG) immune response against the bacteria to indicate PD outcomes in this newly developed ETSPPI mouse model. Our study provides insight into the sex-specific miRNA expression profile of bacterial infection. In addition, this study highlights the basis and rationale for including both sexes as potential biomarkers in the study of global miRNA expression patterns in periodontitis.

ETSPPI-Induced PAHMM Colonization in C57BL/6J Mice
Nine-week-old male and female mice were randomly divided into groups for ETSPPI (n = 10) and sham-infection (n = 10) ( Table 1). Analysis of oral gingival plaque samples from each mouse after bacterial infection [colony polymerase chain reaction (PCR)] showed the presence of bacterial 16S rRNA gene amplicons in agarose gel electrophoresis. Both sexes of mice infected with the early ecological colonizer S. gordonii showed the presence of S. gordonii-specific 16S rRNA gene amplicons (100%) after the first infection cycle, indicating that all mice were colonized with Gram+ S. gordonii. Previous studies showed that this is a critical ecological event for the adherence or coaggregation of the late colonizer bacterium P. gingivalis with the initial colonizer S. gordonii supragingival, and P. gingivalis enters the subgingival cavity to multiply [18,19]. However, the intermediate colonizer F. nucleatum (found in 50% of the male mice and 70% of the female mice) was colonized after its first infection cycle, and 100% colonization was observed after the second infection cycle. Similarly, F. nucleatum colonization is critical because it bridges with numerous Gram+ supragingival and Gram− subgingival late colonizers (e.g., P. gingivalis, T. denticola, and T. forsythia). Subsequently, after 3 weeks of infection, polymicrobial infection with P. gingivalis, T. denticola, and T. forsythia showed colonization on the gingival surfaces of all mice (100%) ( Table 2). None of the sham-infected (male or female) mice were positive at any point for genomic DNA of any of the five bacteria analyzed. These results confirmed the colonization of the five oral bacteria and the development of the PAHMM mouse model.  Periodontitis was examined using morphometry by measuring the horizontal ABR. Both male and female mice infected with PAHMM had a higher ABR in the mandible (lingual) (p < 0.0001 for male mice; adjusted p-value = 0.0001 for female mice) and maxilla (palatal) (p < 0.0001 for male and female mice). Similarly, a higher ABR was also observed in the maxilla (buccal) in both male and female mice (adjusted p-value = 0.0001; Figure 1B,C). Accordingly, there were no differences in ABR (periodontitis outcome measure) between male and female ETSPPI mice. Serum from all mice was evaluated for the humoral IgG immune response against the formalin-killed (FK) whole-cell antigens of S. gordonii, F. nucleatum, P. gingivalis, T. denticola, and T. forsythia. The IgG immune response was higher in female mice against P. gingivalis (> 10 4 -fold; p < 0.001), T. forsythia (> 10 4 -fold; p < 0.001), and T. denticola (> 10 2 -fold; p < 0.01), whereas a higher IgG immune response was observed only against P. gingivalis (> 10 4 -fold; p < 0.001) in male mice ( Figure 1D). No IgG antibody response was observed against S. gordonii or F. nucleatum in either male or female infected mice. Bacteria-specific genomic DNA from all five oral microbes was identified in the heart after ETSPPI (Table 3) using PCR (S. gordonii DNA in male (9/10), female (4/10); F. nucleatum DNA in male (9/10), female (3/10); P. gingivalis DNA in male (4/10), female (2/10); T. denticola DNA in male (10/10), female (0/10); T. forsythia DNA in male (9/10), female (5/10)). This finding suggests a synergistic interaction leading to the physiological colonization/infection of the gingival epithelium and the intravascular dissemination of bacteria to the distal organs. All five bacteria disseminated from the oral gingival surface to the heart, representing their invasive potential. Specifically, intravascular dissemination was higher in the heart tissue in males than females, and this was significant for F. nucleatum and T. denticola (* p < 0.05, Fisher's exact test). Furthermore, there was significantly higher dissemination of all bacteria except T. denticola in the lungs of females than males (* p < 0.05, Fisher's exact test) ( Table 3). S. gordonii DNA was present in other internal organs, including the lungs, kidney, and liver. DNA of all five bacteria was more prevalent in the lungs of female mice than male (Table 3).
The dissemination of all five bacteria from the oral gingival surface to the heart represents their invasive potential. Intravascular dissemination to heart tissue was higher in males than females, which was significant for F. nucleatum and T. denticola (* p < 0.05, Fisher's exact test). There was significantly higher dissemination of all bacteria except T. denticola in the lungs of females compared to males (* p < 0.05, Fisher's exact test).

Nanostring Analysis of miRNAs in Bacteria-Infected Mandibles
The nucleic acid purity (260/280 and 260/230 ratio) of total RNA isolated from both bacteria-infected and sham-infected (both sexes) mouse mandibles (n = 6) was above 2, which was high enough to proceed with sample preparation in the nCounter miRNA assay (Table S1). The expression levels of spike-ins and positive and negative controls were similar in all the samples that showed ligation. Hybridization was efficient in all the mandible samples during sample preparation ( Figure S1A). A heatmap representation showed the DE of miRNAs between male and female mice in bacteria-infected and sham-infected groups ( Figure 2A). The multidimensional scaling plot also showed sample clustering and the similarity of miRNA expression in each sample. It is one of the most unique graphical representations of data to show the relationship between the samples. Samples with similar miRNA expressions were clustered together, and samples with dissimilar miRNA expression were segregated. PAHMM and ETSPPI male and female mice showed the DE of miRNA between samples ( Figure 2B).

Differentially Expressed miRNAs
nCounter miRNA expression profiling showed 190 DE miRNAs in male infected mice and 188 DE miRNAs in female infected mice ( Figure 2C). Among 378 DE miRNAs, miRNAs that showed > 1-fold difference with a p-value between 0.9 to 0.0001 were used for data analysis. The number of DE miRNAs in both sexes infected with bacteria was not significantly different. The Venn diagram of the upregulated miRNAs between male and female bacteria-infected mice shows unique upregulated miRNAs in male (34.2%) and female (46.1%) mice. In both sexes of bacteria-infected mice, 19.7% of the miRNAs were commonly upregulated ( Figure 2D). Similarly, the Venn diagram of the downregulated miRNAs between male and female bacteria-infected mice shows unique downregulated miRNAs in male (41.3%) and female (32%) mice. In both sexes of bacteria-infected mice, 26.7% of the miRNAs were commonly downregulated ( Figure 2E). Further analysis with a higher fold difference from 1.1 to 2.0 and a p FDR -value of < 0.1 showed highly significant DE of miRNA in both male and female infected mice. The volcano plot depicts three upregulated miRNAs (miR-1274a, miR-377, and miR-690) and seven downregulated miRNAs in male infected mice (miR-382, miR-434-3p, miR-411, miR-miR-369-3p, miR-LET-7f, miR-146a, and miR-376c) ( Figures 3A and 4A). Similarly, four upregulated (miR-9, miR-148a, miR-669a, and miR-199a-3p) and three downregulated miRNAs (miR-206, miR-193b, and miR-69) were observed in female infected mice ( Figures 3B and 4B). These DE miRNAs were unique; no common miRNAs were found between male and female mice. No significant difference was observed in major inflammatory miRNAs such as miRNA-155, miR-132, or miR-146a expression in bacteria-and sham-infected mice, nor between sexes ( Figure S1B).   Y-axis. The black dots represent the miRNAs that do not pass the filter parameters. Three significant upregulated miRNAs and seven significant downregulated miRNAs are shown in the table. (B) The volcano plot depicts the upregulated (green) and downregulated (purple) miRNAs that showed a fold difference of ±1.1 with pFDR of < 0.1. The log ratio of the fold change is on the X-axis, and the negative log of the pFDR is on the Y-axis. The black dots represent the miRNAs that do not pass the filter parameters. Four upregulated miRNAs and three downregulated miRNAs are shown in the table.

Discussion
We investigated the sex-specific levels of the colonization/infection of five bacteria, the microbe-induced IgG immune antibody response, intravascular dissemination, and ABR after infection. All five bacteria were colonized in the gingival surface of the mice, which we confirmed through 16S rRNA gene-specific amplification. As expected, both sexes of mice that were infected by ETSPPI had a significantly higher maxillary and mandibular ABR. This finding provides direct evidence that all mice infected with ETSPPI that mimics PAHMM exhibited periodontitis because ABR is considered one of the outcome measurements of chronic periodontitis. The bone resorption measurement results are consistent with previously published studies in which polybacterial infection in several mouse models (e.g., ApoE −/− , itgβ6, TLR2 −/− , and TLR4 −/− ) induced higher bone resorption [21,[23][24][25][26]. No significant difference in ABR was observed between male and female mice infected with ETSPPI.
In contrast, there were sex-specific differences in the genomic DNA of all five bacteria detected in heart tissue. The intravascular dissemination of all five bacteria from gingival tissues to the heart indicates a synergistic interaction of early, intermediate, and late colonizers that adhered to the gingiva after ETSPPI. Furthermore, the observed in vivo male-specific dissemination to the heart directly correlates with the Atherodent clinical trial, in which the association between PD and unstable angina was more common in men, with an average age of 54 years [27]. In addition, a recent study conducted in Spain identified a higher prevalence of cardiovascular disease (CVD) among patients with PD, which was almost double in men [28]. It is important to observe that genomic DNA of all five bacteria was detected in human aortic atherosclerotic plaque and CVD [29]. We previously demonstrated that P. gingivalis, T. denticola, T. forsythia, and F. nucleatum induced atherosclerotic aortic plaque in an ApoE −/− model of atherosclerosis [25,26]. Further data indicate that S. gordonii has a highly invasive potential to disseminate from gingival epithelial tissues to distal organs such as the lungs, kidney, and liver and to gain access to systemic circulation. We also observed a similar dissemination pattern of S. gordonii to the heart, lungs, spleen, and kidneys in both APP-transgenic CRND8 (Tg) and wild-type nontransgenic (ntg) mice [30]. In addition, S. gordonii is not only a commensal supragingival bacterium but also an opportunistic pathogen, and it can enter the bloodstream, leading to endocarditis [31]. It can also cause apical periodontitis or caries and exists on the heart valves of patients with infective endocarditis [32][33][34][35].
The female-specific intravascular dissemination of the five bacteria to the lungs was surprising. Oral microbiota settling down in the oral microecosystem is known as the main source of the lung microbiome, and it is strongly associated with the occurrence and development of several respiratory diseases (e.g., pneumonia, chronic obstructive pulmonary disease (COPD), lung cancer, cystic fibrosis lung disease, asthma) [36]. In addition, poor oral health and periodontal bacterial dysbiosis (i.e., aspiration of oral microbes and interactions between oral microbes and respiratory pathogens) are related to the risk of multiple respiratory diseases or lung infection. A recent review supports a possible association between poor periodontal health, the frequency of exacerbations, hospitalization, and quality of life in patients with COPD [37].
Significant serum IgG antibody levels of P. gingivalis, T. denticola, and T. forsythia in female mice were observed, whereas in male mice, no significant serum IgG antibody levels were observed for T. denticola or T. forsythia. These in vivo data clearly suggest that these bacteria can synergistically alter the host's immune competence with the increased production of virulence factors (i.e., P. gingivalis virulence factors decrease the host response) by subverting innate-immune signaling [38]. Thus, a sex-specific difference in the induction of immunity correlates with sex-specific differences in the oral microbiome and the clinical severity and incidence of PD among women and men [15]. High-throughput miRNA profiling documented 190 DE miRNAs in male mandibles and 188 DE miRNAs in female mandibles. The DE miRNAs in male and female mice were unique. Although ABR and IgG antibody levels in both male and female mice were similar, but the miRNA expression profiles in the mandibles of male and female mice were unique (76%), and only < 24% of the miRNAs were common. This suggests that miRNA expression in the ETSPPI model with PAHMM is based on sex as a biological variable. A homogenous sex group generates more consistent data than a mixed-sex one; most of our previous studies used male mice for experimental periodontitis. Our previous study with three major periodontal pathogens, namely P. gingivalis, T. denticola, and T. forsythia, in ApoE −/− male mice showed an enhanced expression of miR-146a in the maxilla and spleen, which was considered a dominant inflammatory miRNA [2]. However, the ETSPPI used in this study with five different bacterial infections did not show the enhanced expression of miR-146a, miR-132, or miR-155. Multiple explanations may have contributed to these different miRNA's observations, such as the use of a different mouse model, the change in infection schedule, the addition of two bacteria (S. gordonii and F. nucleatum), the mouse-infected mandible tissue miRNA, and most importantly the use of high-throughput nCounter ® miRNA Expression Panels.
We reported three (miR-1274a, miR-377, and miR-690) and four (miR-9, miR-148a, miR-669a, and miR-199A-3p) upregulated miRNAs in male and female mice, respectively. This is the first report to show the presence of these upregulated miRNAs in the PAHMM mouse model. Among the seven upregulated miRNAs, only miR-9 [1,22] and miR-148a [6] have been reported to be upregulated in inflamed gingival tissues in humans. The other miRNAs (miR-199a-3p, miR-1247a, miR-377, miR-690, and miR-669a) identified in this study have not been previously evaluated in patients with periodontitis. Hence, further studies using high-throughput techniques are warranted to identify global miRNAs in inflamed human gingival tissues. miR-9 has been shown to classically activate macrophages and various pro-inflammatory molecules, and it plays an important role in regulating the inflammatory response [39]. On the other hand, miR-148a is also closely associated with the MAPK signaling pathway and the NF-kB pathway, an important pathway in inflammation and the immune system [40,41]. Hence, our observation of a higher expression of miR-9 and miR-148a in the ETSPPI mouse model supports the concept that these miRNAs are important regulators of an inflammatory response in experimental PD. Furthermore, miR-9 and miR-148a have been closely associated with breast, gastric, lung, and colorectal cancers in humans [42][43][44][45][46][47][48].
miR-199a-3p has been reported to be a tumor-suppressor miRNA in lung cancer proliferation [49], and miR-1274a has been reported as an oncogene and prognostic marker for colon cancer [50] and non-small-cell lung cancer [51]. Further studies with miRNA knockout mice may reveal other possible functions of miR-199a-3p and miR-1274a in experimental periodontitis. The male-specific miRNA-377, which was significantly (2-fold) upregulated only in male mice, also regulates cerebral inflammation and angiogenesis in ischemic stroke [52]. Interestingly, several observational, cross-sectional [53,54], casecontrol [55,56], and cohort studies [57,58] determined an association between PD and stroke incidence that strongly supports miRNA-377 as a direct link between microbe-induced gingival and cerebral inflammation. A strong positive relationship was found between miR-377 upregulation in males and the genomic dissemination of all five bacteria to male heart tissue. Furthermore, miR-377 has been linked with other inflammatory diseases, such as atherosclerosis [59] and Sjogren's syndrome [60], indicating a strong miRNA link between atherosclerosis and periodontitis. miRNA-690 has been implicated in inflammation and endoplasmic reticulum stress in obesity [61]. Further studies are warranted to determine whether miR-377 and miRNA-690 play an important role in proinflammatory cytokine responses in periodontal bacteria-mediated inflammation. A detailed comparison analysis of the presence of important miRNAs identified in this study in human periodontitis and other diseases is given in Table 4.

Bacterial Strains, Growth Conditions, and Polybacterial Preparations
S. gordonii DL1, F. nucleatum ATCC 49256, P. gingivalis 381, T. denticola ATCC 35405, and T. forsythia ATCC 43037 were used in this study. P. gingivalis 381, S. gordonii DL1, and F. nucleatum ATCC 49256 were grown in Brucella blood agar plates supplemented with hemin and vitamin K (Hardy Diagnostics, Santa Maria, CA, USA). The oral spirochete T. denticola ATCC 35405 strain was grown in GM-1 broth [2,21], and T. forsythia ATCC 49307 was grown in TSB broth supplemented with N-acetyl muramic acid (5 mg/mL) and hemin (1 mg/mL) [21,63]. All the bacteria were cultured and harvested in a Coy anaerobic chamber at 37 • C for 2 to 3 days as previously described [24,25,30,63]. P. gingivalis, S. gordonii, and F. nucleatum were harvested from the media plates by using a sterile cotton tip applicator. The log-phase culture of T. denticola and T. forsythia was harvested by centrifugation at 8000 rpm for 10 min, and the respective pellets were washed with 5 mL of phosphate-buffered saline (PBS) followed by suspension in 1 mL of reduced transport fluid (RTF). An equal volume of P. gingivalis and T. denticola in RTF was taken in round bottom culture tubes and incubated for 5 min inside an anaerobic chamber (Hausser Scientific Company, Horsham, PA, USA). After incubation, T. forsythia was added to the culture tubes, and it was allowed to interact for an additional 5 min. Bacterial cells were counted using a Petroff-Hausser bacterial counting chamber. The three bacterial mixtures (10 8 cells each) were then mixed with an equal volume of 6% carboxymethylcellulose (CMC), and polybacterial infection was performed. An equal volume of RTF and 6% CMC was used as a vehicle control for sham-infected mice.

Ecological Time-Sequential Polybacterial Periodontal Infection in C57BL/6J Mice
Subgingival microbial dysbiosis in the periodontium leads to the progression of inflammatory periodontitis. Periodontal microbes colonize in supragingival and subgingival regions and activate various proinflammatory cytokines and chemokines, leading to the breakdown of connective tissues and this causes ABR. Understanding the host-pathogen synergistic interaction during the progression of inflammation and identifying the key miR-NAs involved in inflammatory reactions is important for designing prognostic/diagnostic markers for inflammatory PD. Because the prevalence of chronic periodontitis differs between men and women, it is imperative to identify whether miRNA expression also varies between sexes in experimental PD. To partially mimic the normal bacterial colonization that occurs in the human oral cavity from youth to adulthood, we used an ETSPPI model in C57BL/6J mice.
Both female and male C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Mice were housed in microisolator cages and fed standard chow and sterile water ad libitum. All mouse cages were maintained at 25 • C with alternating 12-h periods of light and dark. To avoid cross-contamination, sham-infected mice were maintained in separate rooms. All animal procedures were done in accordance with the approved protocol guidelines from the University of Florida Institutional Animal Care and Use Committee (IACUC protocol #201910761). Nine-week-old male and female mice were randomly divided into groups for polybacterial infection (n = 10) and sham infection (n = 10) ( Table 1). After acclimation, mice were initially administrated kanamycin (500 mg/kg) in sterile water for 3 d followed by swabbing with 0.12% chlorhexidine gluconate (Peridex: 3M ESPE Dental Products, St. Paul, MN, USA) mouth rinse on day 5. This initial treatment suppresses existing oral bacteria and facilitates the bacterial colonization with S. gordonii, F. nucleatum, P. gingivalis, T. denticola, and T. forsythia. To mimic the human ecological bacterial colonization, ETSPPI was performed. Polybacterial-infected mice were first infected with S. gordonii (an early colonizer; 2.5 × 10 8 cells) for 2 infection cycles (1 infection cycle constitutes 4 consecutive infection days in a week), followed by infection with F. nucleatum (an intermediate colonizer, bridging bacterium; 2.5 × 10 8 cells) for 2 infection cycles. Finally, this was followed by infection with P. gingivalis, T. denticola, and T. forsythia (late colonizers; 2.5 × 10 8 cells each) for 6 infection cycles. Because PD is associated with polybacterial synergy and dysbiosis of the oral bacterium, ETSPPI in the current study with PAHMM (early, intermediate, and late bacterial colonizers) in mice partially mimics the ecological bacterial shifts from youth to adulthood in humans.
Sequential polybacterial infection was performed as illustrated in Figure 1A. Mouse gingival surfaces were swabbed with a sterile cotton swab every alternate infection week, and 16S rRNA gene-specific PCR for specific bacteria was performed to monitor bacterial adhesion/colonization and the subsequent invasion of the gingival epithelium. At the end of the 20-week infection period, mice were euthanized. Right maxilla and mandibles were collected for bone morphometry analysis. Left maxilla and mandibles were stored in RNAlater (Invitrogen, Waltham, MA, USA). Left mandibles (periodontium) with three molars were used for Nanostring analysis. Blood was drawn through cardiac puncture, and serum was isolated and stored in a −20 • C freezer for measuring IgG antibodies against specific bacteria.

Bacterial Genomic DNA Detection in Gingival Plaque Using PCR
After 4 days of infection, gingival plaque samples from the gingival surface were collected from all infected and sham-infected mice by using sterile cotton swabs. Bacterial genomic DNA was detected using 16S rRNA gene species-specific primers of the bacteria using Phusion High Fidelity Master Mix from New England Biolabs (NEB, Ipswich, MA, USA) as described previously [21,25,26,64].

Bacterial Systemic Dissemination to Distal Organs
Genomic DNA from an aliquot of the heart comprising the right atrium and right ventricle and other organs such as the lungs, kidney, and liver was extracted following a standard protocol described in the Qiagen Dneasy Blood and Tissue kit (Qiagen, Germantown, MD, USA). 16S rRNA gene-specific PCR was performed for the respective bacterium using specific bacterial primers as described [21,25,26,64].

Antibacterial Serum Immunoglobulin G Antibody Analysis by Enzyme-Linked Immunosorbent Assay
A serum IgG antibody titer against each of the five bacterial species from all the mice in the four groups was analyzed by using enzyme-linked immunosorbent assay (ELISA) as described previously [21,25,26,64]. Whole cell FK bacteria such as S. gordonii (1:60 dilution), F. nucleatum (1:50), P. gingivalis (1:150), T. denticola (1:30), and T. forsythia (1:160) were used as coating antigen, and 100 µL of 1:100 diluted serum of each mouse in triplicate was added to the wells. The plates were incubated for 2 h. One hundred microliters of goat anti-mouse IgG alkaline phosphatase (Sigma Aldrich, St. Louis, MO, USA) was added, incubated, and developed with 200 µL of p-nitrophenylphosphate for 15 min, and color development was stopped using 3M NaOH. The plates were read at OD 405 nm and analyzed using Gen5 software in Epoch Microplate Spectrophotometer (BioTek, Winooski, VT, USA). IgG serum antibody concentrations were determined by using the gravimetric standard curve (Sigma Aldrich).

Alveolar Bone Resorption by Morphometry
The horizontal ABR area of infected male and female mice was measured by histomorphometry as described previously [21,25,26,64]. The right mandibles and maxilla were autoclaved, and the defleshed jaws were immersed in 3% hydrogen peroxide for 30 min and air-dried. Two-dimensional molar teeth images were captured using a stereo dissecting microscope (Stereo Discovery V8, Carl Zeiss Microimaging, Inc, Thornwood, NY, USA). The area between the cemento-enamel junction to the alveolar bone crest of the buccal and the palatal surfaces of the maxillary jaws was measured by using the line tool (AxioVision LE 29A software version 4.6.3, Thornwood, NY, USA). Two examiners blinded to the study mice groups measured the ABR.

Extraction and Quantification of RNA for Nanostring Analysis
Mandibles (left; n = 6) from male and female mice (four groups) were taken for Nanostring analysis. The total RNA from each mandible was extracted based on the protocol described in the mirVana miRNA isolation kit (Ambion, Austin, TX, USA). Initially, mandibles from each mouse were entirely homogenized using the handheld rotor-stator homogenizer with sterile individual TissueRuptor disposable probes (Qiagen; Germantown, MD, USA) for each sample. After homogenization, each sample was lysed in a denaturing lysis solution that stabilized RNAs and inactivated RNases. The samples were subjected to an acid-phenol:chloroform extraction that removed all the cellular components such as protein, DNA, and other cellular products. The aqueous phase was removed, transferred to the fresh nuclease-free microcentrifuge tube, and 1.25 volume of 100% ethanol was added. This mixture was transferred to the filter cartridge placed into the collection tube and centrifuged for 15 s at 10,000× g. The filter cartridge was washed with 700 µL of wash solution-1 followed by washing with wash solution-2/3. After washing, the total RNA was eluted from the filter cartridge with 100 µL of nuclease-free water. RNA yield and purity were determined using a Take3 micro-volume plate in Epoch Microplate Spectrophotometer (BioTek), and quantification was performed in technical duplicates for each sample.

nCounter ® miRNA Expression Profiling
We used high-throughput nCounter ® miRNA Expression Panels (Nanostring Technologies, Seattle, WA, USA) to effectively identify the DE of miRNA in both sexes. Nanos-tring nCounter ® analysis can identify 577 miRNAs in any sample, and by using molecular barcodes, it can detect even a low number of miRNAs without the need for reverse transcription or amplification. miRNA expression profiling was performed using the Nanostring nCounter ® Mouse miRNA Assay kit v1.5 (Nanostring Technologies). This assay is a highly sensitive multiplexed method that detects miRNAs using molecular barcodes called nCounter reporter probes without the need for reverse transcription. Sample preparation involved annealing, ligation, and purification. These steps were carried out based on the experimental procedure described in the nCounter ® miRNA assay panel kit. Briefly, the annealing master mix was prepared by combining 13 µL of annealing buffer, 26 µL of nCounter miRNA Tag reagent, and 6.5 of diluted (1:500) miRNA assay controls. Next, 3.5 µL of the annealing master mix was aliquoted into each tube of the strip tube and 100 ng of the total RNA from six left mandibles from each group (maximum 3-µL volume) with 260/280 and 260/230 ratios of greater than 2 was added to the respective tubes. The strip tube was placed in the Thermal Cycler with the following conditions: 94 • C for 1 min, 65 • C for 1 min, 45 • C for 1 min, and 48 • C for hold.
After annealing, 2.5 µL of the ligation master mix (19.5 µL of polyethylene glycol (PEG) and 13 µL of ligation buffer) was added to all the tubes in the strip tube. The strip tube was incubated at 48 • C for 5 min, followed by the addition of 1 µL of ligase into each tube without removing the strip tubes from the Thermal Cycler. Ligation was performed with the following conditions: 48 • C for 3 min, 47 • C for 3 min, 46 • C for 3 min, 45 • C for 3 min, 65 • C for 10 min, and 4 • C for hold. To remove the unligated tags, a purification step was performed after adding 1 µL of ligation cleanup enzyme to all the tubes and incubating the tubes at 37 • C for 1 h, 70 • C for 10 min, and 4 • C for hold. Forty microliters of RNase-free water were added to each tube in the strip tube and the sample was ready for hybridization with the nCounter reporter and capture probes. After denaturation at 85 • C for 5 min, a 5-µL aliquot from the miRNA sample preparation tube was taken along with the 10 µL of miRNA reporter code, 10 µL of hybridization buffer, and 5 µL miRNA capture probe. The strip tubes were incubated at 65 • C for 18 h in the Thermal cycler, and the samples were immediately processed for post-hybridization with the nCounter analysis system at the Molecular Pathology Core at the University of Florida.
The nCounter ® Mouse miRNA Assay kit v1.5 provided six positive hybridization controls and eight negative control probes to monitor hybridization efficiency. All components and reagents needed for sample preparation at the reparation station were taken from the nCounter master kit (Nanostring Technologies). Twelve samples per cartridge were processed in a single run, which took 3 h. This was followed by digital analysis, which involved the transfer of the cartridge to the multichannel epifluorescence digital analyzer. A cartridge definition file with a maximum fields of view (FOV) count of 555 per flow cell was taken for digital analysis. The number of images taken per scan corresponded to the number of immobilized reporter probes on the cartridge. A separate Reporter Code Count (RCC) file for each sample containing the count for each probe was downloaded and used for data analysis.

Nanostring Data Analysis
Initial data analysis was performed using nSolver 4.0. Initially, RCC files were imported into nSolver and imaging quality control (QC), binding density QC, positive control linearity QC, and positive control limit of detection QC were carried out as recommended in the system QC parameters. The lanes were flagged when the percent FOV registration was less than 75% for imaging QC. Binding density was outside the range from 0.1 to 2.25 for binding density QC, and the positive control R 2 value was less than 0.95. All 24 samples passed the QC, and no flag lanes were observed. Raw data were generated after passing the QC. To reduce the background signal/noise, the background threshold count value was set to 52 and calculated by taking an average of eight negative control probe counts from all 24 samples. Codeset content normalization parameters were cho-sen, and the normalization was performed based on the top 100 miRNA genes expressed. The normalized factor was calculated based on the geometric mean values of the miRNA genes expressed in each sample. All the normalized data were further analyzed using ROSALIND ® with a hyperscale architecture developed by ROSALIND Inc. (San Diego, CA, USA). Fold changes in the genes were calculated based on the ratio of the difference in the means of the log-transformed normalized data to the square root of the sum of the variances of the samples in the two groups. The limma R library [65] was used to calculate fold changes. Clustering of miRNA for the final heatmap of DE miRNA was done using the Partitioning Around Medoids method with the fpc R library.

Statistical Analysis
One-way ANOVA with Dunnett's multiple comparisons was performed for multiple group comparisons to determine the statistical significance, using the statistical software Prism 9.2.0 (GraphPad Software, San Diego, CA, USA). All the data in graphs are presented as mean ± SEM. p FDR -value < 0.1 was considered statistically significant, and the p FDR -values were calculated based on the Benjamini-Hochberg procedure [66]. The Mann-Whitney test was used for IgG antibody analysis. Two-tailed t-testing was performed on the log-transformed normalized data that assumed unequal variance to identify the differential gene expression. The distribution of the t-statistics was calculated using the Welch-Satterthwaite equation for the degrees of freedom to estimate the 95% confidence intervals for the identified DE of miRNA between groups. Fisher's exact tests were performed to compare the frequencies of bacterial 16S rRNA gene detection in the heart and lungs of experimental male and female mice. Analysis was performed using STATA software version 14.0 (StataCorp, College Station, TX, USA). p < 0.05 was considered significant.